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Abstract

Objective—Interindividual variation in pathways affecting cellular cholesterol metabolism can influence levels of plasma cholesterol, a well-established risk factor for cardiovascular disease. Inherent variation among immortalized lymphoblastoid cell lines from different donors can be leveraged to discover novel genes that modulate cellular cholesterol metabolism. The objective of this study was to identify novel genes that regulate cholesterol metabolism by testing for evidence of correlated gene expression with cellular levels of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) mRNA, a marker for cellular cholesterol homeostasis, in a large panel of lymphoblastoid cell lines.

Approach and Results—Expression array profiling was performed on 480 lymphoblastoid cell lines established from participants of the Cholesterol and Pharmacogenetics (CAP) statin clinical trial, and transcripts were tested for evidence of correlated expression with HMGCR as a marker of intracellular cholesterol homeostasis. Of these, transmembrane protein 55b (TMEM55B) showed the strongest correlation (r=0.29; P=4.0E−08) of all genes not previously implicated in cholesterol metabolism and was found to be sterol regulated. TMEM55B knockdown in human hepatoma cell lines promoted the decay rate of the low-density lipoprotein receptor, reduced cell surface low-density lipoprotein receptor protein, impaired low-density lipoprotein uptake, and reduced intracellular cholesterol.

Conclusions—Here, we report identification of TMEM55B as a novel regulator of cellular cholesterol metabolism through the combination of gene expression profiling and functional studies. The findings highlight the value of an integrated genomic approach for identifying genes that influence cholesterol homeostasis.

Introduction

Immortalized lymphoblastoid cell lines (LCLs), created by Epstein–Barr virus transformation of peripheral blood mononuclear cells,1 have been used as a model system to study genetic variation affecting cholesterol metabolism and statin response. LCLs were first used within the context of familial hypercholesterolemia to functionally assess the effects of specific familial hypercholesterolemia mutations on low-density lipoprotein receptor (LDLR) cell surface protein and rates of LDL-uptake.2,3 In addition, we have previously performed cellular phenotyping of LCLs to identify the functional effects of genetic variation within 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) and LDLR associated with variation in plasma LDL-cholesterol both at baseline and in response to statin treatment.4–7

More recently, studies of LCLs have moved beyond functional analysis of specific gene variants and have proven to be informative for discovery-based purposes as well. For example, given the unclear genetic background of familial combined hyperlipidemia, expression array analysis of LCLs from patients with familial combined hyperlipidemia versus healthy controls was used to identify disease-specific transcriptomic profiles.8 Through expression array analysis of 480 in vitro simvastatin and sham incubated LCLs derived from participants of the Cholesterol and Pharmacogenetics (CAP) statin clinical trial, we recently reported the identification of RHOA as a novel candidate gene implicated in LDL-cholesterol lowering in response to statin treatment.9 Because HMGCR encodes the rate-limiting enzyme of the cholesterol biosynthesis pathway and is tightly regulated at the level of gene transcription by sterol response element binding factor 2 (SREBF2) in response to changes in intracellular sterol content,10 we identified genes whose statin-induced expression level changes were most highly correlated with statin-induced changes in HMGCR.

Here, we sought to determine whether we could extend that line of reasoning to cells in the untreated state to identify novel genes not previously implicated in cholesterol metabolism, hypothesizing that interindividual variation in cellular HMGCR transcript levels could serve as a marker of genetic regulation of cellular cholesterol homeostasis. Using gene expression array data from a set of 480 LCLs from participants of the CAP clinical trial, we tested transcriptome-wide for genes with evidence of correlated expression with HMGCR. From this analysis, transmembrane protein 55b (TMEM55B) emerged as the top novel candidate gene not previously implicated in cholesterol metabolism. Genetic manipulation of TMEM55B in hepatoma cell lines demonstrated that this gene is a novel regulator of cellular cholesterol metabolism that modulates LDLR levels and activity.

Materials and Methods

Materials and Methods are available in the online-only Supplement.

Results

Identification of TMEM55B as a Candidate Gene Involved in Cholesterol Metabolism

To discover novel candidate genes involved in cellular cholesterol metabolism, we sought to identify genes whose expression levels were correlated with HMGCR transcript levels using genome-wide gene expression data from 480 LCLs derived from participants of the CAP clinical trial. From this analysis, 110 genes were identified to have correlated expression with HMGCR, Bonferroni-adjusted P<0.05 (Table; Table II in the online-only Data Supplement for complete list). As expected, the genes most highly correlated with HMGCR transcript levels were those encoding enzymes within the cholesterol biosynthesis pathway and LDLR. From this list, transmembrane protein 55b (TMEM55B) was identified to be the gene most highly correlated (r=0.29; Bonferroni-adjusted P=4.0E−08) with HMGCR transcript levels that has not been previously implicated in cholesterol metabolism.

TMEM55B Is a Novel Sterol-Regulated SREBF Target Gene

To test whether endogenous TMEM55B transcript levels were changed in response to intracellular sterol content, we treated 3 human hepatoma cell lines, HepG2, Hep3B, and Huh7, to conditions of extreme sterol depletion using media containing 2 μmol/L simvastatin+10% lipoprotein-deficient serum (LPDS), thus reducing both cholesterol synthesis and uptake. After 24 hours, we added back sterols using either LDL-cholesterol or 25-hydroxycholesterol and subsequently quantified gene expression. LDLR gene expression, used as a positive control to verify incubation conditions, demonstrated the expected increase with sterol depletion and decrease with add-back. Similarly, extreme sterol depletion (statin+LPDS) induced TMEM55B transcript levels 1.7- to 2.8-fold depending on cell type, whereas sterol add-back reversed this effect (Figure 1A). Induction of TMEM55B transcript levels with statin treatment alone was also observed after in vitro simvastatin exposure (0.5 and 2.0 μmol/L) of primary human hepatocytes derived from 4 unrelated donors (Figure 1B). Because LDLR transcript level was not increased in a statistically significant manner across both statin concentrations, HMGCR transcript was quantified as a second positive control to validate the exposure conditions. Finally, quantification of hepatic TMEM55B in African Green monkeys before and after a 19-week 0.6 mg/kcal cholesterol supplemented diet found that cholesterol loading reduced TMEM55B transcript levels ≈40% (n=5 animals; P=0.01; Figure 1C). We observed similar results in an independent study of African Green monkeys fed a diet containing 0.002, 0.2, or 0.4 mg/kcal cholesterol for 10 weeks (n=5 per diet; Figure 1D), with cholesterol loading downregulating TMEM55B transcript levels in a dose-dependent manner. Similar to results observed in vitro, cholesterol-induced changes in TMEM55B were approximately the same magnitude as those observed in LDLR transcript levels. Furthermore, interindividual variation in hepatic TMEM55B expression levels was directly correlated to variation in hepatic total cholesterol, free cholesterol, and cholesterol esters (Figures 1E; Figure IA and IB in the online-only Data Supplement), whereas there was no relationship between variation in hepatic LDLR transcript levels with hepatic cholesterol (r2=0.01 for total cholesterol). These results support the likelihood that interindividual variation in hepatic cholesterol in response to cholesterol feeding is mediated, in part, by the variation in TMEM55B expression levels.

Sterol regulation of TMEM55B transcript levels was not limited to liver-derived tissues because TMEM55B transcript levels were also upregulated in simvastatin versus sham-treated CAP LCLs after 24 hours (1.2±0.01-fold; n=480; false discovery rate, <0.0001). TMEM55B induction was also observed when sterol-depletion was performed by alternative means than statin treatment and in additional cell models. For instance, in HeLa cells and human fibroblasts cultured in LPDS and exposed to the cholesterol-reducing agent 2-hydroxyl-β-cyclodextrin, TMEM55B transcript was upregulated by 2.4±0.7-fold (n=4; P=0.13) and 3.4±0.6-fold (n=3; P=0.05), respectively.

Transcript levels of genes involved in the maintenance of intracellular cholesterol levels are primarily regulated by the sterol-dependent transcription factors SREBF1 and SREBF2 (aka SREBP1 and 2).10 We queried publicly available SREBF2 ChIP-Seq data sets,11 and found evidence for SREBF2-binding motifs in the promoter region of the TMEM55B gene (chr14:20 929 629–20 930 186; GRCh37/hg19) in both HepG2 and a human immortalized LCL (Figure IB in the online-only Data Supplement). To confirm SREBF2 regulation of TMEM55B gene expression, we quantified TMEM55B in Huh7 cells transfected with siRNAs targeting SREBF1, SREBF2, both SREBF1+SREBF2, or a nontargeting control (NTC). Both SREBF1 and 2 knockdown reduced TMEM55B transcript levels ≈30% (P=0.03 and P=0.04, respectively) with no additive effect of the dual knockdown (Figure IC in the online-only Data Supplement). Similar results were observed in HeLa cells, where SREBF2 knockdown suppressed sterol-dependent upregulation of TMEM55B (Figure ID in the online-only Data Supplement).

TMEM55B knockdown modulates low-density lipoprotein receptor (LDLR). HepG2, Huh7, or Hep3B cells were transfected with siRNAs specific for TMEM55B (TMEM-1 or TMEM-2) or a nontargeting control (NTC) for 48 hours. Where indicated, HepG2 cells were transfected with either an empty vector or a TMEM55B expression plasmid. A, TMEM55B transcript levels were quantified by quantitative polymerase chain reaction (n=8 per cell line and siRNA). B, HeLa cells expressing TMEM55B-GFP together with either TMEM55B siRNA or control siRNA were quantified for total cell-associated GFP-signal. Data were obtained from 300 to 400 cells from 10 background-subtracted images per condition (n=4 experiments). One set of representative images are shown. C, Intracellular cholesterol was quantified by the Amplex Red Cholesterol Assay Kit in HepG2 (n=8) and Huh7 (n=6) cells. D, HepG2 cells (n=3) were transfected with TMEM-1 or NTC siRNAs for 48 hours, after which cycloheximide was added to inhibit protein synthesis. Aliquots were collected after 3 hours, and total LDLR protein was quantified by Western blot with values normalized to β-actin. Values shown are log transformed and expressed relative to time 0. E, LDLR protein was quantified after TMEM55B knockdown in HepG2 (n=12) and Huh7 cells (n=12) or TMEM55B overexpression (HepG2, n=6) via fluorescence-activated cell sorter as previously described.4F, LDL uptake was quantified in HepG2 cells (n=14) and Hep3B cells (n=12) after TMEM55B knockdown or in HepG2 cells after TMEM55B overexpression (n=6) using DiI-labeled LDL as previously described.4 Paired 2-tailed t tests were used to calculate statistically significant differences in gene expression between NTC and TMEM55B siRNA transfected cells or empty vector and TMEM55B expression plasmid transfected cells. TMEM55B indicates transmembrane protein 55b.

Intracellular cholesterol levels are regulated, in part, by cholesterol uptake via LDLR.12 Because TMEM55B has been shown to localize to the endosome/lysosome,13 and LDLR recycles between this compartment and the cell surface,12 we hypothesized that TMEM55B could modulate LDLR turnover, activity, or localization. To determine the potential effect of TMEM55B on LDLR protein, we first transfected HepG2 cells with either siRNA targeting TMEM55B or a nontargeting control siRNA for 48 hours, then treated the cells with cycloheximide, an inhibitor of protein synthesis, and finally collected samples after 3 hours. Although we did not observe detectable degradation of total cellular LDLR protein in the NTC-treated cells, TMEM55B knockdown caused a reduction in LDLR protein after 3 hours, consistent with an accelerated LDLR protein decay rate (Figure 2D; Figure IIIB in the online-only Data Supplement).

Next, we analyzed whether TMEM55B knockdown reduced cell surface LDLR protein. As shown in Figure 2E, cell surface LDLR was reduced in HepG2 cells transfected with either TMEM-1 (0.87±0.05-fold; n=12; P=0.02) or TMEM-2 (0.79±0.05-fold; n=12; P=0.001), as well as in a second hepatoma cell line, Huh7, transfected with TMEM-2 (0.75±0.08-fold; n=12; P=0.02). Consistent with the reduction in cell surface LDLR protein, we also found that TMEM55B knockdown reduced LDL uptake in HepG2 cells transfected with either TMEM-1 (0.92±0.02; n=20; P=0.004) or TMEM-2 (0.92±0.02; n=20; P=0.001), as well as in Hep3B cells transfected with either TMEM-1 (0.95±0.02; n=12; P=0.03) or TMEM-2 (0.94±0.02; n=12; P=0.01; Figure 2F). Overexpression of TMEM55B in HepG2 cells produced the opposite effects, with increases of both cell surface LDLR protein and DiI-LDL uptake relative to control transfected cells (Figure 2E and 2F). We found no evidence that TMEM55B knockdown influenced either levels of cell surface transferrin receptor or rate of transferrin-568 uptake (Figure IIIC and IIID in the online-only Data Supplement). Finally, we found that TMEM55B knockdown with either TMEM-1 or TMEM-2 in both HepG2 and Huh7 cells did not reduce LDLR transcript levels but rather caused a 20% to 50% increase under all conditions tested (Figure IV in the online-only Data Supplement). Notably, TMEM55B knockdown did not generate consistent changes in transcript levels of other SREBF2 target genes, such as HMGCR or PCSK9 (Figure IV in the online-only Data Supplement). These results support the hypothesis that TMEM55B modulates LDLR cell surface protein levels through a post-transcriptional process.

Discussion

Here, we have used genome-wide gene expression level results from LCLs to identify candidate loci associated with HMGCR transcript levels, which serve as a biomarker for variation in cellular cholesterol metabolism, from which TMEM55B emerged as a novel candidate gene associated with cellular cholesterol metabolism. Recently, we have successfully used a similar approach to identify RHOA as a novel determinant of LDL-cholesterol response to statin treatment.9 Although cellular phenotyping of LCLs has long been used to functionalize the molecular effect of genetic variation associated with elevated cholesterol levels,2–4,6,9 to our knowledge this is the first instance of capitalizing on the interindividual variation of a cellular phenotype within LCLs to identify novel genes involved in cellular cholesterol homeostasis.

TMEM55B, also known as type I phosphatidylinositol 4,5-bisphosphate 4-phosphatase, was first identified as an enzyme that catalyzes the hydrolysis of 4-position phosphate on phosphatidylinositol-(4,5)-bisphosphate (PtdIns-4,5-P2 aka PIP2) but not other phosphatidylinositides, to generate phosphatidylinositol-5-phosphate (PtdIns-5-P or PI(5)P).13 Phosphatidylinositides control the timing and localization of endocytic membrane trafficking by recruiting components of the transport machinery, thus regulating intracellular membrane traffic. It has been well established that PIP2, the predominant phosphatidylinositide formed at the plasma membrane, is required for clathrin-mediated endocytosis, with roles in clathrin-coated pit initiation, stabilization, and maturation.14–17 Because one of the major mechanisms for cholesterol uptake is through clathrin-mediated endocytosis of LDL-bound LDLR, it is possible that TMEM55B modulates LDLR activity through the regulation of phosphatidylinositides.

In support of that hypothesis, here we show that TMEM55B knockdown stimulated LDLR protein decay, resulting in reduced cell surface LDLR protein and LDL uptake. The increased LDL uptake seen with TMEM55B overexpression was consistent with positive correlation noted between interindividual variation in hepatic TMEM55B transcript levels and hepatic total cholesterol from 2 independent experiments of cholesterol-fed African green monkeys (greater TMEM55B transcript leads to greater LDL cholesterol uptake, which leads to greater hepatic cholesterol). Importantly, we did not observe a relationship between variation in LDLR transcript levels and hepatic cholesterol, suggesting that the relationship observed is not because of variation in sterol-induced changes in TMEM55B transcript levels. Thus, these results support the likelihood that variation in hepatic TMEM55B may directly modulate cholesterol metabolism.

TMEM55B may also modulate LDLR regulation through a nonclathrin-mediated mechanism. For example, the F-actin network structure, which impedes LDLR trafficking by creation of a physical barrier to vesicle movement, has also been shown to be dependent on PIP2 concentrations.18 In addition, PI(5)P has been hypothesized to play a role in exocytosis from late endosome compartments and mediate TMEM55B effects on EGF-receptor decay rates.19,20 Because nascent LDLR is transported to the plasma membrane through the endoplasmic reticulum-Golgi pathway, and mature LDLR is known to recycle between the plasma membrane and endosome/lysosomes, it is possible that TMEM55B effects on phosphatidylinositides other than PIP2 may affect LDLR intracellular trafficking. The importance of proper intracellular trafficking/signaling and sorting of LDLR has been recently shown by Zeigerer et al,21 who reported that hepatic Rab5 knockdown in mice caused a ≈10-fold increase in plasma LDL levels when compared with controls because of a reduction of the entire endolysosomal system (early and late endosomes and lysosomes), increasing the number of clathrin-coated pits and vesicles and reducing the rate of LDL uptake. Additional studies are necessary to determine the precise mechanism by which TMEM55B regulates LDLR.

Notably there are ≥40 kinases and phosphatases that affect phosphatidylinositide metabolism; however, TMEM55B seems to be the only gene in this pathway that is sterol responsive (Elizabeth Theusch, et al, personal communication, 2014). Furthermore, although TMEM55B might be expected to affect trafficking through the endolysosomal system generally, we did not observe an effect of TMEM55B knockdown on either levels of cell surface transferrin receptor or amount of 568-labeled transferrin within endosome-like particles, suggesting that LDLR is particularly sensitive to the effects of TMEM55B.

Although our studies strongly support the likelihood that TMEM55B directly modulates LDLR, interestingly we found that TMEM55B knockdown reduced intracellular cholesterol (total, free, and esters) between 30% and 60% although the reduction in LDLR cell surface protein and LDL uptake was closer to 10%. This discrepancy suggests that TMEM55B may modify other pathways that influence cellular cholesterol metabolism, such as cholesterol efflux or synthesis. A key component of the insulin-signaling cascade is the activation of phosphoinositide 3-kinase (PI3-kinase) that phosphorylates PIP2 to produce phosphatidylinositol (3,4,5)-triphosphate (aka PIP3). Notably, VLDL synthesis and secretion are regulated by insulin. Cellular models have shown that generation of PIP3 is necessary for insulin-mediated suppression of VLDL,22 whereas inhibition of PIP3 generation in vivo enhances hepatic VLDL apolipoprotein B secretion.23,24 Because both TMEM55B and PI3-kinase share PIP2 as a substrate, it is possible that TMEM55B knockdown affects intracellular cholesterol levels through changes in VLDL metabolism. Similarly, activation of phosphatidylinositol-specific phospholipase C, an enzyme that also catalyzes the hydrolysis of PIP2, has been previously linked to high-density lipoprotein–induced cholesterol efflux.25 Thus, additional studies are necessary to determine the precise mechanism(s) by which TMEM55B influences cholesterol metabolism.

In summary, using a novel approach of testing for evidence of correlated expression with HMGCR, we have identified TMEM55B as a novel gene that influences cholesterol homeostasis. We have validated this finding by functional studies demonstrating that TMEM55B regulates cellular levels of cholesterol and LDLR. Because increased LDLR activity is a major determinant of the clinical efficacy of statins and other drugs used for the prevention and treatment of cardiovascular disease, these results have implications for the development of novel drug targets.

Sources of Funding

This work was supported by National Institutes of Health National Heart, Lung, and Blood Institute R01 HL104133 (Dr Medina), U19 HL069757 (Dr Krauss), PO1-HL049373 (Dr Rudel), R00 HL088528 (Dr Temel), American Heart Association Fellowship 12POST10430005 (Dr Theusch) as well as a Career Development Award (12CDA04) and the Transatlantic Networks of Excellence in Cardiovascular Research Program (10CVD03) of the Foundation Leducq (Dr Runz).

Significance

Activity of the low-density lipoprotein receptor (LDLR) is a major determinant of circulating levels of LDL-cholesterol, a well-established risk factor of cardiovascular disease. Here, we report identification of TMEM55B as a novel regulator of both LDLR and cellular cholesterol metabolism using a combination of gene expression profiling and functional studies. Nascent LDLR is transported to the plasma membrane through the endoplasmic reticulum-Golgi pathway, and mature LDLR is known to recycle between the plasma membrane and the endosome/lysosomes. Transmembrane protein 55b catalyzes the conversion of phosphatidylinositides, membrane-bound signaling molecules that control the timing and localization of intracellular trafficking, suggesting that transmembrane protein 55b regulates LDLR by modulating its intracellular movement. Greater understanding of the molecular mechanisms guiding LDLR recycling may be useful for not only improving our understanding of the pathways underlying interindividual variation in LDL-cholesterol but also informing the development of novel drugs for the treatment and prevention of cardiovascular disease.